EP2615103B1 - Method for detecting adenocorticotropic hormone and absorbent - Google Patents
Method for detecting adenocorticotropic hormone and absorbent Download PDFInfo
- Publication number
- EP2615103B1 EP2615103B1 EP20130150698 EP13150698A EP2615103B1 EP 2615103 B1 EP2615103 B1 EP 2615103B1 EP 20130150698 EP20130150698 EP 20130150698 EP 13150698 A EP13150698 A EP 13150698A EP 2615103 B1 EP2615103 B1 EP 2615103B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- acth
- gelled casein
- gelled
- carrier
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims description 58
- 229940088597 hormone Drugs 0.000 title description 4
- 239000005556 hormone Substances 0.000 title description 4
- 230000002745 absorbent Effects 0.000 title 1
- 239000002250 absorbent Substances 0.000 title 1
- 239000005018 casein Substances 0.000 claims description 307
- 235000021240 caseins Nutrition 0.000 claims description 304
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 303
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 claims description 112
- 229960000258 corticotropin Drugs 0.000 claims description 109
- 239000000275 Adrenocorticotropic Hormone Substances 0.000 claims description 108
- 101800000414 Corticotropin Proteins 0.000 claims description 108
- 102400000739 Corticotropin Human genes 0.000 claims description 108
- 239000007788 liquid Substances 0.000 claims description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 33
- 210000002700 urine Anatomy 0.000 claims description 22
- 210000002966 serum Anatomy 0.000 claims description 21
- 239000003463 adsorbent Substances 0.000 claims description 20
- 108010088751 Albumins Proteins 0.000 claims description 16
- 102000009027 Albumins Human genes 0.000 claims description 16
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 16
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical group Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 15
- 239000004971 Cross linker Substances 0.000 claims description 10
- 239000012736 aqueous medium Substances 0.000 claims description 10
- 150000001413 amino acids Chemical group 0.000 claims description 9
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 210000002381 plasma Anatomy 0.000 claims description 3
- 102000011632 Caseins Human genes 0.000 description 293
- 108010076119 Caseins Proteins 0.000 description 293
- ZOEFCCMDUURGSE-SQKVDDBVSA-N cosyntropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 ZOEFCCMDUURGSE-SQKVDDBVSA-N 0.000 description 123
- 108010091893 Cosyntropin Proteins 0.000 description 116
- 239000011259 mixed solution Substances 0.000 description 100
- 239000000243 solution Substances 0.000 description 98
- 239000000523 sample Substances 0.000 description 88
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 75
- 239000006228 supernatant Substances 0.000 description 71
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 66
- 238000002360 preparation method Methods 0.000 description 46
- 229910021642 ultra pure water Inorganic materials 0.000 description 45
- 239000012498 ultrapure water Substances 0.000 description 45
- 239000006185 dispersion Substances 0.000 description 43
- 239000000499 gel Substances 0.000 description 38
- 239000007853 buffer solution Substances 0.000 description 28
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 28
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 25
- 239000007998 bicine buffer Substances 0.000 description 25
- 238000001179 sorption measurement Methods 0.000 description 22
- 238000011084 recovery Methods 0.000 description 21
- 239000000203 mixture Substances 0.000 description 18
- 238000001514 detection method Methods 0.000 description 17
- 239000002244 precipitate Substances 0.000 description 17
- 238000001962 electrophoresis Methods 0.000 description 16
- 239000012723 sample buffer Substances 0.000 description 14
- 238000005406 washing Methods 0.000 description 14
- 238000005259 measurement Methods 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 108010010803 Gelatin Proteins 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 239000008273 gelatin Substances 0.000 description 11
- 229920000159 gelatin Polymers 0.000 description 11
- 235000019322 gelatine Nutrition 0.000 description 11
- 235000011852 gelatine desserts Nutrition 0.000 description 11
- 239000013074 reference sample Substances 0.000 description 11
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 10
- 239000011734 sodium Substances 0.000 description 10
- 229910052708 sodium Inorganic materials 0.000 description 10
- 239000013068 control sample Substances 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 6
- 229910052709 silver Inorganic materials 0.000 description 6
- 239000004332 silver Substances 0.000 description 6
- 239000012472 biological sample Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 208000002109 Argyria Diseases 0.000 description 4
- 108010049003 Fibrinogen Proteins 0.000 description 4
- 102000008946 Fibrinogen Human genes 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 101000609413 Homo sapiens Inter-alpha-trypsin inhibitor heavy chain H4 Proteins 0.000 description 4
- 102100039457 Inter-alpha-trypsin inhibitor heavy chain H4 Human genes 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 229940012444 factor xiii Drugs 0.000 description 4
- 229940012952 fibrinogen Drugs 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000012146 running buffer Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 239000006173 Good's buffer Substances 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 230000003139 buffering effect Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- 108010071289 Factor XIII Proteins 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 206010021067 Hypopituitarism Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 239000011542 SDS running buffer Substances 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003997 corticotropin derivative Substances 0.000 description 2
- 238000005115 demineralization Methods 0.000 description 2
- 230000002328 demineralizing effect Effects 0.000 description 2
- 239000003398 denaturant Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- AIDBEARHLBRLMO-UHFFFAOYSA-M sodium;dodecyl sulfate;2-morpholin-4-ylethanesulfonic acid Chemical compound [Na+].OS(=O)(=O)CCN1CCOCC1.CCCCCCCCCCCCOS([O-])(=O)=O AIDBEARHLBRLMO-UHFFFAOYSA-M 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 239000000055 Corticotropin-Releasing Hormone Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000251124 Mustelus canis Species 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010036297 Postpartum hypopituitarism Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 201000009895 Sheehan syndrome Diseases 0.000 description 1
- 241000271567 Struthioniformes Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 239000003470 adrenal cortex hormone Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 229940064004 antiseptic throat preparations Drugs 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical class NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000960 hypophysis hormone Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 201000009958 panhypopituitarism Diseases 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
- 235000021246 κ-casein Nutrition 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/665—Assays involving proteins derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- G01N2333/695—Corticotropin [ACTH]
Definitions
- the present invention relates to a method for detecting adrenocorticotropic hormone (ACTH) and an adsorbent of ACTH and uses thereof.
- ACTH adrenocorticotropic hormone
- peptides are contained in the blood.
- peptides having a blood concentration different from the usual blood concentration in the living body in a certain disease state are attracting attention as markers for disease in the field of clinical tests.
- Simmonds' disease and Sheehan's syndrome caused by the reduced secretion of pituitary hormone can be clinically tested by detecting ACTH as a marker peptide.
- impurities contained in a biological sample to be used for the clinical test may have an influence on the detection of ACTH. Consequently, there is a demand for a method for detecting ACTH which can suppress the influence of impurities in the biological sample.
- the present inventors have found out that a gel obtained from casein can selectively adsorb ACTH and completed the present invention.
- the present invention provides:
- the gelled casein contained in the adsorbent of ACTH of the present invention can selectively adsorb ACTH in the liquid sample. Therefore, the method for detecting ACTH of the present invention can detect ACTH in a state where influences of other impurities are limited by allowing ACTH in the liquid sample to be selectively adsorbed to the gelled casein.
- detection method a process of bringing a liquid sample containing ACTH into contact with gelled casein to allow ACTH to be adsorbed to the gelled casein is performed.
- ACTH (a hormone composed of 39 amino acids) is secreted from the pituitary gland in the brain and acts on the adrenal cortex to facilitate secretion of adrenocortical hormone.
- the ACTH being detected may be a natural ACTH produced in the living body, a peptide produced by cells such as mammalian cells into which the gene coding for ACTH is introduced, insect cells, Escherichia coli, or a peptide produced by chemical synthesis.
- the ACTH has a high homology in amino acid sequence between biological species. Accordingly, the origin of ACTH is not particularly limited as long as it is a biological species that produces ACTH. Examples thereof include mammals (e.g., humans, mice, rats, dogs, and rabbits), birds (e.g., ostriches), and fish (e.g., smooth dogfish).
- the liquid sample is not particularly limited as long as it is a liquid sample which may contain ACTH.
- it may be a biological sample.
- the biological sample includes body fluid.
- Preferable examples of the body fluid include blood, plasma, serum, and urine.
- the sample may contain a substance capable of binding to ACTH, which is derived from the living body.
- ACTH in the liquid sample may form a complex with the substance capable of binding to ACTH.
- the substance which can be bound to ACTH includes albumin.
- the ACTH contained in the liquid sample may be the entire length of polypeptide or a fragment thereof.
- a polypeptide having at least an amino acid sequence from the 1st to 24th position of ACTH is preferred.
- casein means a phosphorus protein which is known as a main component of milk protein. It is known that the casein is composed of three major casein components: ⁇ s , ⁇ , and ⁇ .
- casein which is used as a raw material of the gelled casein is not particularly limited.
- examples thereof include acid casein, casein sodium, ⁇ s -casein, ⁇ -casein, and ⁇ -casein.
- the origin of casein is not particularly limited. Usable examples thereof include casein obtained from the milk of mammals such as cows and goats.
- the method for preparing gelled casein is not particularly limited as long as it can gelate casein.
- it includes a method for preparing gelled casein by reacting a solution of casein with a crosslinker.
- a crosslinker water-soluble carbodiimide (WSC, chemical name: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferable.
- the gelled casein can be obtained, for example, in the following manner. First, 0.1 to 20% (w/v) of a casein solution is mixed with 0.1 to 50% (w/v) of a WSC solution at a volume ratio of 1 : 0.2 to 50. The mixed solution thus obtained is heated at 65 to 200°C for 5 to 120 minutes to return to room temperature, resulting in formation of gelled casein in the mixed solution. Then, the gelled casein is recovered from the mixed solution by centrifugation, followed by washing with water or an appropriate buffer solution to obtain gelled casein.
- the form of the gelled casein is not particularly limited. Accordingly, the gelled casein obtained in the above manner may be suitably processed into a particle form, a membranous form or the like.
- the operation of bringing the gelled casein into contact with the liquid sample is not particularly limited.
- the gelled casein may be added to the liquid sample to bring them into contact with each other, or a dispersion of the particulate gelled casein may be mixed with the liquid sample to bring them into contact with each other.
- the gelled casein is preferably brought into contact with the liquid sample under the conditions where the pH in the mixture of the gelled casein and the liquid sample is 6.5 or more, preferably from 6.5 to 10.
- the pH in the mixture of the gelled casein and the liquid sample is 6.5 or more, preferably from 6.5 to 10.
- a unit configured to adjust the pH is not particularly limited and it can be suitably selected from methods known in the art.
- the pH can be adjusted by previously adding a buffer having a buffering action at a pH of 6.5 or more to the gelled casein and/or the liquid sample or adding the buffer to a mixture of the gelled casein and the liquid sample.
- the content ratio of the gelled casein to the liquid sample is from 1 : 2 to 1 : 1000 (at a volume ratio), preferably from 1 : 10 to 1 : 200.
- the ambient temperature is from 4 to 60°C, preferably from 4°C to room temperature, and the contact time is from 5 seconds to 60 minutes, preferably from 30 seconds to 1 minute.
- the process of detecting the ACTH adsorbed to the gelled casein is performed after the contact of the gelled casein with the liquid sample.
- a unit configured to detect the ACTH adsorbed to the gelled casein is not particularly limited.
- the gelled casein adsorbed to ACTH is recovered so that the ACTH adsorbed to the recovered gelled casein can be directly detected.
- the gelled casein adsorbed to ACTH is recovered and the ACTH is allowed to be released from the recovered gelled casein so that the released ACTH can be detected. That is, the detection method of the present invention may further include recovering the gelled casein brought into contact with the liquid sample and allowing the ACTH adsorbed to the recovered gelled casein to be released.
- a unit configured to recover the gelled casein may be suitably selected from units known in the art according to the form of the gelled casein used.
- the gelled casein can be recovered by centrifuging the mixture of the gelled casein and the liquid sample and removing the supernatant.
- a unit configured to allow ACTH to be released from the gelled casein is not particularly limited.
- ACTH can be eluted from the gelled casein by adding any protein denaturant known in the art to the gelled casein recovered.
- protein denaturant include surfactants such as sodium dodecyl sulfate (SDS), urea, and guanidine.
- the present inventors have found the fact that the affinity of the gelled casein for ACTH varies depending on the pH. That is, in the embodiment of the present invention, the contact of the recovered gelled casein with an aqueous medium at a pH of 5.5 or less allows the ACTH adsorbed to the gelled casein to be released into the aqueous medium.
- a unit configured to adjust the pH is not particularly limited as long as it is a unit which does not prevent the subsequent detection of ACTH. It can be suitably selected from methods known in the art.
- the aqueous medium is not particularly limited.
- an aqueous medium which can adjust the pH in the mixture of the gelled casein and the aqueous medium to 5.5 or less.
- the aqueous medium include inorganic acids known in the art (e.g., dilute hydrochloric acid and dilute sulfuric acid), organic acids (e.g., acetic acid solutions), and buffer solutions having a buffering action at a pH of 5.5 or less (e.g., phosphate buffers and Good's buffers).
- a unit configured to detect the released ACTH is not particularly limited as long as it is a unit capable of detecting ACTH by distinguishing from other substances. It can be suitably selected from peptide detection methods known in the art. Examples of the detection unit include SDS-PAGE, subsequent gel staining or Western blotting, ELISA, chromatography, and mass spectrometry.
- the detection method of the present invention can be used when intending to selectively recover ACTH from a liquid sample and/or when intending to purify ACTH from a liquid sample. Therefore, the scope of the present invention includes a method for selectively recovering ACTH from a liquid sample and a method for purifying ACTH from a liquid sample.
- the adsorbent of ACTH for use according to the method of the present invention has a feature of containing the gelled casein and can be suitably used for the detection method of the present invention. That is, the ACTH in the liquid sample can be selectively adsorbed by using the adsorbent of the present invention, similarly to the gelled casein described in the detection method of the present invention.
- the ACTH to be adsorbed may be a polypeptide having at least an amino acid sequence from the 1st to 24th position of ACTH. Further, the ACTH may form a complex with a substance capable of binding to ACTH such as albumin.
- the form of the gelled casein contained in the adsorbent for use in the method of the present invention is not particularly limited. From the viewpoint of easy handling, the particle form is preferred.
- the adsorbent may be in the form of a dispersion prepared by dispersing in water or an appropriate buffer solution.
- the adsorbent may be in the form of a biuret reagent containing gelled casein particles dried by a known method such as freeze drying and water or an appropriate buffer solution for dispersing the particles at the time of usage.
- the concentration of the gelled casein particles in the dispersion can be suitably set by those skilled in the art. Usually, it is from 1 to 100 mg/ml, preferably from 5 to 50 mg/ml.
- the adsorbent for use in the method of the present invention may contain known additives in the art, in addition to the gelled casein.
- the additives include buffer solutions (e.g., phosphate buffers and Good's buffers), protein stabilizing agents (e.g., BSA), and antiseptics (e.g., sodium azide).
- buffer solutions e.g., phosphate buffers and Good's buffers
- protein stabilizing agents e.g., BSA
- antiseptics e.g., sodium azide
- the affinity of the gelled casein for ACTH changes according to the pH as described above.
- the method for storing the gelled casein contained in the adsorbent for use in the method of the present invention is not particularly limited, and it may be stored at, for example, 4°C to room temperature.
- the adsorbent for use in the method of the present invention has a capacity of selectively adsorbing to ACTH as long as it contains the gelled casein produced in the same manner as described in the detection method of the present invention.
- the capacity can be confirmed by the detection method of the present invention.
- the adsorption capacity of ACTH may be confirmed as follows. First, a synthesized ACTH peptide solution with a known concentration is mixed with the produced adsorbent. The gelled casein contained in the adsorbent is recovered from the mixed solution thus obtained. The concentration of the synthesized ACTH peptide contained in the remaining solution is measured and the resulting concentration is compared with the concentration before mixing. As the result of comparison, if the concentration after mixing is sufficiently lower than that before mixing, it is determined that the produced adsorbent has a capacity as the adsorbent of the present invention.
- Casein sodium (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in MilliQ (registered trademark) water (hereinafter simply referred to as "ultrapure water”; Millipore) to prepare a casein sodium solution (50 g/L (5%)).
- a 5% water-soluble carbodiimide (WSC, manufactured by DOJINDO LABORATORIES) solution (1 mL) and ultrapure water (3 mL) were added to the casein sodium solution (1 mL) to prepare a casein sodium-WSC mixed solution (5 mL) (final concentration of casein sodium: 1%, final concentration of WSC: 1%).
- the mixed solution was heated at 80°C for 10 minutes, followed by returning to room temperature to form a gel in the mixed solution.
- the supernatant was removed with a pipette from the solution containing the gel and the gelled casein was collected.
- Ultrapure water was added to the collected gelled casein.
- the mixture was centrifuged at 22200 G for 10 minutes using a centrifuge (CF15RXII, manufactured by Hitachi Koki Co., Ltd.) to allow it to be separated into a supernatant and gelled casein, followed by removal of the supernatant with pipette.
- the operation was repeated twice to wash the gelled casein.
- the gelled casein after washing was made into a fine gel carrier by vortexing and pipetting and the resulting carrier was dispersed in ultrapure water (5 mL).
- the obtained dispersion was a 1% gelled casein carrier dispersion.
- bovine serum albumin manufactured by Sigma Aldrich
- Gelatin manufactured by Wako Pure Chemical Industries, Ltd.
- a 10% gelatin solution 0.5 mL
- the solution was cooled at 4°C for 2 hours to obtain a gelatin gel.
- Ultrapure water (4.5 mL) at 4°C was added to the obtained gelatin gel and the mixture was made into a fine gelatin gel carrier by pipetting. Then, a 1% gelatin gel carrier dispersion was prepared.
- the 1% gelled casein carrier dispersion (50 ⁇ L) prepared in (1) was mixed with a solution of a polypeptide having at least an amino acid sequence from the 1st to 24th position of ACTH (hereinafter referred to as "ACTH (1-24)" (300 ⁇ M, 5 ⁇ L, manufactured by Biologica Co.) which was labeled with tetramethyl rhodamine (TMR).
- ACTH (1-24) 300 ⁇ M, 5 ⁇ L, manufactured by Biologica Co.
- TMR tetramethyl rhodamine
- a 1 M Bicine/NaOH buffer solution (pH 9) (10 ⁇ L) and ultrapure water (35 ⁇ L) were added to the mixture to prepare a mixed solution (100 ⁇ L).
- the final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of TMR-labeled ACTH (1-24) was adjusted to 15 ⁇ M.
- the 1% albumin gel carrier dispersion in place of the 1% gelled casein carrier dispersion and the 1% gelatin gel carrier dispersion were used to prepare each mixed solution. The operation of the gelatin gel carrier was performed while cooling so that the gelatin gel was not dissolved in the solution.
- the gelled casein carrier had a high adsorption capacity to TMR-labeled ACTH (1-24) as compared with those of the albumin gel carrier and the gelatin gel carrier.
- Example 2 Evaluation 1 of selectivity of adsorption capacity of gelled casein carrier to ACTH (1-24)
- the 1% gelled casein carrier dispersion (50 ⁇ L) prepared in Example 1 (1), a 1 M Bicine buffer solution (pH 9) (3 ⁇ L), and ultrapure water (7 ⁇ L) were added to the mixture to prepare a peptide mixed solution (100 ⁇ L).
- the final concentration of the gelled casein carrier in the peptide mixed solution was adjusted to 0.5% and the final concentration of each peptide was adjusted to 100 ⁇ M.
- the peptide mixed solution thus prepared was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate, and the supernatant was collected.
- Ultrapure water 99 ⁇ L was added to the collected supernatant (1 ⁇ L) to prepare a 100-fold diluted supernatant.
- a 1 ⁇ M TMR-labeled ACTH (1-24) solution 1 ⁇ L was added to the supernatant to prepare a sample for measuring the peptide adsorption capacity.
- a control sample was prepared by the same operation as described above using ultrapure water in place of the 1% gelled casein carrier dispersion.
- ⁇ CHCA solution 10 mg/mL was prepared as a matrix solution.
- a solution of 0.1% trifluoroacetic acid/50% acetonitrile was used as a solvent.
- Drops of the sample for measuring the peptide adsorption capacity (1 ⁇ L) prepared in (1) were put on an MS sample plate and dried under reduced pressure. Thereafter, drops of the matrix solution (1 ⁇ L) were put on the plate to be dried. It was confirmed that matrix crystals were formed.
- the MALDI-TOF/MS measurement m/z of 800 to 5000
- Voyager-DE (trademark) PRO manufactured by Applied Biosystems
- the MALDI-TOF/MS measurement of the control sample was performed by the same operation as that of the MALDI-MS of the sample for measuring the peptide adsorption capacity using the control sample in place of the sample for measuring the peptide adsorption capacity. These measurement results were adjusted by the strength of the index sample to calculate the concentration of the peptides contained in the supernatants of each peptide.
- the results of the adsorption capacity of the gelled casein carrier to ACTH (1-24), ITIH4 fragment, Fibrinogen ⁇ fragment, and FactorXIII fragment are shown in Fig. 2 .
- the properties of the peptides are shown in Table 1. In Fig.
- Example 3 Evaluation 2 of selectivity of adsorption capacity to ACTH (1-24) of gelled casein carrier
- a 1% gelled casein carrier dispersion was prepared in the same manner as described in (1) Preparation of gelled casein carrier dispersion of Example 1.
- the 1% gelled casein carrier dispersion (50 ⁇ L) prepared in (1) was mixed with a 300 ⁇ M TMR-labeled ACTH (1-24) solution (10 ⁇ L). Further, a 1 M Bicine buffer solution (pH 9) (10 ⁇ L) and ultrapure water (30 ⁇ L) were added thereto to prepare a mixed solution (100 ⁇ L). The final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of TMR-labeled ACTH (1-24) was adjusted to 30 ⁇ M. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate.
- a mixed solution for comparison was also prepared by the same operation as described above using ultrapure water in place of the 1% gelled casein carrier dispersion.
- the fluorescence intensity of the supernatant at an excitation wavelength of 540 nm and a fluorescence wavelength of 580 nm in the mixed solution of the gelled casein carrier and TMR-labeled ACTH (1-24) prepared in (2) was measured with the spectrophotofluorometer (F-7000, manufactured by Hitachi High-Technologies Corporation.).
- the fluorescence intensity of the mixed solution not containing the gelled casein carrier prepared in (1) as a control was measured.
- the fluorescence intensity of the control was converted at 30 ⁇ M TMR-labeled ACTH (1-24) to calculate the amount of the TMR-labeled ACTH (1-24) which was not adsorbed to the gelled casein carrier and left in the supernatant.
- the fluorescence intensity of the supernatant at an excitation wavelength of 540 nm and a fluorescence wavelength of 580 nm in the mixed solution of the gelled casein carrier and TMR prepared in (2) was measured with the spectrophotofluorometer (F-7000, manufactured by Hitachi High-Technologies Corporation.).
- the fluorescence intensity of the mixed solution not containing the gelled casein carrier prepared in (1) as a control was measured.
- the fluorescence intensity of the control was converted at 30 ⁇ M TMR to calculate the amount of the TMR which was not adsorbed to the gelled casein carrier and left in the supernatant. Those results are shown in Fig. 3 .
- a 1 M Bicine buffer solution (pH 9) (10 ⁇ L) and ultrapure water (40 ⁇ L) were added to the 1% gelled casein carrier dispersion (50 ⁇ L) prepared in Example 1 to prepare a mixed solution (100 ⁇ L).
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate. The resulting precipitate was collected as a sample B.
- the 1% gelled casein carrier dispersion (50 ⁇ L) prepared in Example 1 was mixed with a solution (10 ⁇ L) of 300 ⁇ M TMR-labeled ACTH (1-24).
- a 1 M Bicine buffer solution (pH 9) (10 ⁇ L) and ultrapure water (30 ⁇ L) were added thereto to prepare a mixed solution (100 ⁇ L).
- the final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of ACTH (1-24) was adjusted to 30 ⁇ M.
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate. The supernatant was collected as a sample C.
- the supernatant (10 ⁇ L) obtained by centrifugation was subjected to SDS-PAGE.
- the sample B collected in (1) was subjected to the same operation as that of the sample D and SDS-PAGE.
- a 2-fold condensed sample buffer (5 ⁇ L) was added to 5 ⁇ L of the sample A collected in (1), which was heated at 70°C for 10 minutes. 10 ⁇ L of the resulting mixture was subjected to SDS-PAGE.
- the sample C collected in (1) was subjected to the same operation as that of the sample A and SDS-PAGE.
- electrophoresis apparatus for SDS-PAGE, a vertical mini electrophoresis system (manufactured by Invitrogen) was used.
- electrophoresis gels NuPAGE 4-12% Bis-Tris Gels (1.0 mm, 10 wells, manufactured by Invitrogen) were used.
- As a running buffer a solution prepared by 20-fold dilution of NuPAGE MES SDS running buffer (20x) (manufactured by Invitrogen) was used. The voltage during electrophoresis was set to a constant value of 200 V. After SDS-PAGE, the gel was stained with silver using EzStain Silver (ATTO CORPORATION). The results are shown in Fig. 4 .
- Example 5 Removal of albumin and recovery of ACTH (1-24) from complex to which albumin and ACTH (1-24) are bound (hereinafter referred to as "BSA-ACTH (1-24) complex")
- Casein sodium was dissolved in ultrapure water to prepare a 50 g/L (5%) casein sodium solution.
- a 5% water-soluble carbodiimide (WSC) solution (1 mL) and ultrapure water (3 mL) were added to the casein sodium solution (1 mL) to prepare a casein sodium-WSC mixed solution (5 mL) (final concentration of casein sodium: 1%, final concentration of WSC: 1%).
- the temperature of the mixed solution was returned to room temperature by heating at 80°C for 10 minutes and a gel was formed in the mixed solution.
- the liquid portion was removed from the mixed solution containing the gel with a pipette.
- a gel precipitate was collected as a gelled casein carrier. Ultrapure water was added to the collected gelled casein carrier.
- the resulting mixture was centrifuged at 22200 G for 10 minutes using the centrifuge to allow it to be separated into a supernatant and a gel carrier, followed by removal of the supernatant with a pipette.
- the operation was repeatedly performed twice to wash the gelled casein carrier.
- the gel carrier after washing was made into a fine gel carrier by vortexing and pipetting and the resulting carrier was dispersed in ultrapure water (3 mL).
- the dispersion was a 1.6% gelled casein carrier dispersion.
- a 2400 ⁇ M BSA solution (40 ⁇ L), 200 ⁇ M TMR-labeled ACTH (1-24) (8 ⁇ L), and ultrapure water (32 ⁇ L) were mixed to prepare a BSA-ACTH (1-24) complex solution (80 ⁇ L).
- the concentration of BSA in the solution is 1200 ⁇ M, and the concentration of TMR-labeled ACTH (1-24) was adjusted to 20 ⁇ M.
- the equilibrium dissociation constant (Kd) between BSA and TMR-labeled ACTH (1-24) was measured by the fluorescence titration method and the Kd value was 12 ⁇ M. That is, when the prepared complex solution is 2-fold diluted, the concentration of BSA is 600 ⁇ M.
- the concentration of TMR-labeled ACTH (1-24) is 10 ⁇ M, 98.0% of TMR-labeled ACTH (1-24) forms a complex with BSA.
- the solution (80 ⁇ L) prepared in (2) was mixed with the 1.6% gelled casein carrier dispersion (50 ⁇ L) prepared in (1) . Thereafter, a 1 M Bicine/NaOH buffer solution (pH 9) (16 ⁇ L) and ultrapure water (14 ⁇ L) were added thereto to prepare a mixed solution (160 ⁇ L).
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- the gelled casein carrier was subjected to the following operation (4). The supernatant was used as a "supernatant A" of a sample containing BSA.
- a mixed solution not containing the gelled casein carrier a BSA-ACTH (1-24) complex solution (80 ⁇ L), a 1 M Bicine/NaOH buffer solution (pH 9) (16 ⁇ L), and ultrapure water (64 ⁇ L) were added to prepare a mixed solution (160 ⁇ L) as a control.
- the solution was used as a reference (REF.) sample containing BSA.
- a mixed solution was prepared in the same manner as described in the preparation of mixed solution of gelled casein carrier and BSA-ACTH (1-24) complex using a 20 ⁇ M TMR-labeled ACTH (1-24) solution in place of the BSA-ACTH (1-24) complex solution.
- the gelled casein carrier was subjected to the following operation (4).
- the supernatant was used as a "supernatant A" of a sample not containing BSA.
- a mixed solution not containing the gelled casein carrier 20 mM TMR-labeled ACTH (1-24) (80 ⁇ L), a 1 M Bicine/NaOH buffer solution (pH 9, 16 ⁇ L), and ultrapure water (64 ⁇ L) were added to prepare a mixed solution (160 ⁇ L) as a control.
- the solution was used as a REF. sample not containing BSA.
- a 20 mM Bicine/NaOH buffer solution 160 ⁇ L was added to the gelled casein carrier separated in (3) Preparation of mixed solution of gelled-casein carrier and BSA-ACTH (1-24) complex. The mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the resulting dispersion was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The gelled casein carrier was subjected to the following operation (5). The supernatant was used as a "supernatant B" of a sample containing BSA.
- a 20 mM Bicine/NaOH buffer solution (160 ⁇ L) was added to the gelled casein carrier separated in (3) Preparation of mixed solution of gelled-casein carrier and ACTH (1-24). The mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the resulting dispersion was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The gelled casein carrier was subjected to the following operation (5). The supernatant was used as a "supernatant B" of a sample not containing BSA.
- a 100 mM MES/NaOH buffer solution (pH 5) 160 ⁇ L was added to the gelled casein carrier after the process in (4) Washing of gelled casein carrier mixed with BSA-ACTH (1-24) complex.
- the mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The supernatant was used as a "supernatant C" of a sample containing BSA.
- a 100 mMMES/NaOH buffer solution (pH 5) (160 ⁇ L) was added to the gelled casein carrier after the process in (4) Washing of gelled casein carrier mixed with ACTH (1-24). The mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The supernatant was used as a "supernatant C" of a sample not containing BSA.
- a 10 x Loading Buffer (10 ⁇ L, manufactured by TAKARA), a 60% glycerol solution (5 ⁇ L), and ultrapure water (85 ⁇ L) were mixed to prepare a solution.
- the resulting solution was used as a sample buffer.
- 20 ⁇ L of the REF. sample containing BSA, 20 ⁇ L of the sample supernatants A to C containing BSA, 20 ⁇ L of the REF. sample not containing BSA, and 20 ⁇ L of the sample supernatants A to C not containing BSA were collected.
- Each of the solutions was mixed with the sample buffer (2 ⁇ L), followed by SDS-PAGE.
- electrophoresis apparatus for SDS-PAGE, a vertical mini electrophoresis system (manufactured by Invitrogen) was used.
- electrophoresis gels NuPAGE 4-12% Bis-Tris Gels (1.0 mm, 10 wells, manufactured by Invitrogen) were used.
- As a running buffer a solution prepared by 20-fold dilution of NuPAGE MES SDS running buffer (20x) (manufactured by Invitrogen) was used. The voltage during electrophoresis was set to a constant value of 200 V.
- the bands of TMR-labeled ACTH (1-24) can be confirmed from Fig. 5 .
- the bands of BSA can be confirmed from Fig. 6 .
- the results of the REF. sample of Fig. 6 and the sample (the supernatant A) show that BSA is not adsorbed to the gelled casein carrier in the presence of the Bicine/NaOH buffer solution (pH 9).
- the density of the bands is decreased.
- TMR-ACTH a part of the TMR-ACTH (1-24) is detached from the BSA and adsorbed to the gelled casein carrier. It is surmised that the proportion in which the gelled casein carrier adsorbs TMR-ACTH (1-24) from BSA is determined by the balance between the equilibrium reaction of BSA and ACTH (1-24) and the equilibrium reaction of the gelled casein carrier and ACTH (1-24). In other words, in order to collect a larger amount of TMR-ACTH (1-24) from BSA, it is necessary to increase the additive amount of the gelled casein carrier. In the sample (the supernatant B), the band of TMR-labeled ACTH (1-24) can be hardly confirmed.
- TMR-labeled ACTH (1-24) is still adsorbed to the gelled casein carrier in the washing process.
- the dark bands of TMR-labeled ACTH (1-24) can be confirmed. Accordingly, it was found that the TMR-labeled ACTH (1-24) was released from the gelled casein carrier in the presence of the MES/NaOH buffer solution (pH 5).
- a 1% gelled casein carrier dispersion was prepared in the same manner as described in (1) Preparation of gelled casein carrier dispersion of Example 1.
- the 1% gelled casein carrier dispersion (50 ⁇ L) prepared in (1) was mixed with a 300 ⁇ M TMR-labeled ACTH (1-24) solution (10 ⁇ L), and a 1 M Bicine buffer solution (pH 9) (10 ⁇ L) and ultrapure water (30 ⁇ L) were added thereto to prepare a mixed solution (100 ⁇ L).
- the final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of TMR-labeled ACTH (1-24) was adjusted to 30 ⁇ M.
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- a 20 mM Bicine/NaOH buffer solution (160 ⁇ L) was added to the gelled casein carrier separated in (2), followed by pipetting of the gelled casein carrier. After pipetting, the resulting dispersion was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- a 30 ⁇ M TMR-labeled ACTH (1-24) solution containing each solution with each pH shown in Table 2 and not containing the gelled casein carrier was prepared.
- the fluorescence intensity of the solution was calculated in the same manner as described above.
- the fluorescence intensity of the control was converted at 30 ⁇ M TMR-labeled ACTH (1-24) to calculate the concentration of ACTH (1-24) remained in the supernatant from which the gelled casein carrier was removed.
- the measurement results are shown in Table 2 and Fig. 7 .
- ACTH (1-24) when ACTH (1-24) is detected by a biochemical procedure, a Glycine/NaOH buffer solution which is a Good's buffer or a MES/NaOH buffer solution may be used.
- a Glycine/NaOH buffer solution which is a Good's buffer or a MES/NaOH buffer solution
- ACTH (1-24) can be dissociated using a volatile pH adjuster such as an acetic acid solution.
- a 1% gelled casein carrier dispersion was prepared in the same manner as described in (1) Preparation of gelled casein carrier dispersion of Example 1.
- Serum previously collected from healthy woman (22 years old) (100 ⁇ L, PROMEDDX "Normal Serum Pool") was mixed with 300 ⁇ M TMR-labeled ACTH (1-24) (10 ⁇ L) to prepare serum containing TMR-labeled ACTH (1-24).
- the 1% gelled casein carrier dispersion (100 ⁇ L) prepared in (1) was placed in another container and centrifuged to allow the dispersion to be separated into a supernatant and a precipitate. The supernatant was removed with a pipette and only the precipitate was collected.
- the serum containing TMR-labeled ACTH (1-24) prepared above (110 ⁇ L), a 1 M Bicine buffer solution (pH 9) (20 ⁇ L), and ultrapure water (20 ⁇ L) were added to the obtained precipitate to prepare a mixed solution (150 ⁇ L) of the gelled casein carrier and the sample.
- the final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.75%
- the final concentration of TMR-labeled ACTH (1-24) was adjusted to 20 ⁇ M
- the dilution ratio of the serum was adjusted to 1.5-fold.
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- a mixed solution 150 ⁇ L was prepared by adding serum containing the TMR-labeled ACTH (1-24) (110 ⁇ L), a 1 M Bicine buffer solution (pH 9) (20 ⁇ L), and ultrapure water (20 ⁇ L).
- a gelled casein carrier solution was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and serum containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the serum containing TMR-labeled ACTH (1-24). The solution was used as a control sample A.
- a mixed solution 150 ⁇ L was prepared by adding a 1 M Bicine buffer solution (pH 9) (20 ⁇ L) and ultrapure water (130 ⁇ L).
- a mixed solution of the gelled casein carrier and serum was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and serum containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the TMR-labeled ACTH (1-24).
- the solution was used as a control sample B.
- a mixed solution 150 ⁇ L was prepared by adding serum (100 ⁇ L), a 1 M Bicine buffer solution (pH 9) (20 ⁇ L), and ultrapure water (30 ⁇ L).
- the supernatant separated in (2) Preparation of mixed solution was removed with a pipette and the gelled casein carrier was collected. A 20 mMBicine/NaOH buffer solution (150 ⁇ L) was added to the gelled casein carrier, followed by pipetting of the gelled casein carrier. The resulting solution was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The operation was repeatedly performed twice. The same operation was performed on the controls A and B prepared in (2).
- a 100 mM MES/NaOH buffer solution (pH 5) (50 ⁇ L) was added to the gelled casein carrier after the process of (3) Washing of gelled casein carrier, followed by pipetting of the gelled casein carrier.
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- the supernatant was recovered with a pipette and it was used as a recovered sample. The same operation was performed on the samples of the controls A and B.
- 2-fold condensed sample buffer (5 ⁇ L) was respectively added to the reference sample before recovery for the control samples A and B which were collected in (2), the sample after recovery which was collected in (4), and the sample after recovery for the control samples A and B, followed by heating at 70°C for 10 minutes to prepare samples.
- Each of the obtained samples (1 ⁇ L) was subjected to SDS-PAGE.
- the electrophoresis apparatus for SDS-PAGE, the electrophoresis gels, and the running buffer are the same as those of Example 4 (2).
- the voltage during electrophoresis was set to a constant value of 200 V.
- fluorescence images and silver-stained images were obtained by the same operation as that of Example 5 (6). The results are shown in Fig. 8 .
- a 1% gelled casein carrier fluid concentrate was prepared in the same manner as described in (1) Preparation of gelled casein carrier fluid concentrate of Example 1 (1).
- the urine (110 ⁇ L) containing TMR-labeled ACTH (1-24) prepared above and a 1 M Bicine buffer solution (pH 9) (10 ⁇ L) were added to the obtained precipitate to prepare a mixed solution (120 ⁇ L) of the gelled casein carrier and the sample.
- the final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5%
- the final concentration of TMR-labeled ACTH (1-24) was adjusted to 25 ⁇ M
- the dilution rate of the urine was adjusted to 1.2-fold.
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- a mixed solution 120 ⁇ L was prepared by adding the urine containing TMR-labeled ACTH (1-24) (110 ⁇ L) and a 1 M Bicine buffer solution (pH 9) (10 ⁇ L).
- a gelled casein carrier solution was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and urine containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the urine containing TMR-labeled ACTH (1-24). The solution was used as a control sample A.
- a mixed solution 120 ⁇ L was prepared by adding a 1 M Bicine buffer solution (pH 9) (10 ⁇ L) and ultrapure water (110 ⁇ L).
- a mixed solution of the gelled casein carrier and urine was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and urine containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of TMR-labeled ACTH (1-24).
- the solution was used as a control sample B.
- a mixed solution 120 ⁇ L was prepared by adding urine (100 ⁇ L), a 1 M Bicine buffer solution (pH 9) (10 ⁇ L), and ultrapure water (10 ⁇ L).
- a mixed solution of the gelled casein carrier and TMR-labeled ACTH (1-24) was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and urine containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the urine.
- the solution was used as a control sample C.
- a mixed solution 120 ⁇ L was prepared by adding 300 ⁇ M TMR-labeled ACTH (1-24) (10 ⁇ L), a 1 M Bicine buffer solution (pH 9) (10 ⁇ L), and ultrapure water (100 ⁇ L).
- the supernatant separated in (2) Preparation of mixed solution was removed with a pipette and the gelled casein carrier was collected. A 20 mM Bicine/NaOH buffer solution (120 ⁇ L) was added to the gelled casein carrier, followed by pipetting of the gelled casein carrier. The resulting solution was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The operation was repeatedly performed twice. The same operation was performed on the control samples A to C prepared in (2).
- a 100 mM Glycine/NaOH buffer solution (pH 2.5) (60 ⁇ L) was added to the gelled casein carrier after the process of (3) Washing of gelled casein carrier, followed by pipetting of the gelled casein carrier.
- the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- the supernatant was recovered with a pipette and it was used as a recovered sample. The same operation was performed on the control samples A to C.
- 2-fold condensed sample buffer (5 ⁇ L) was respectively added to the reference sample before recovery for the control samples A to C which were collected in (2), the sample after recovery which was collected in (4), and the sample after recovery for the control samples A to C, followed by heating at 70°C for 10 minutes to prepare samples.
- Each of the obtained samples (10 ⁇ L) was subjected to SDS-PAGE.
- the electrophoresis apparatus for SDS-PAGE, the electrophoresis gels, and the running buffer are the same as those of Example 4 (2).
- the voltage during electrophoresis was set to a constant value of 200 V.
- fluorescence images and silver-stained images were obtained by the same operation as that of Example 5 (6).
- the results are shown in Fig. 9 . From the results of Fig. 9 , it was confirmed that the proteins in the urine were hardly adsorbed to the gelled casein carrier and ACTH (1-24) could be selectively recovered from the urine.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
- The present invention relates to a method for detecting adrenocorticotropic hormone (ACTH) and an adsorbent of ACTH and uses thereof.
- Various peptides are contained in the blood. Among them, there are peptides having a blood concentration different from the usual blood concentration in the living body in a certain disease state. Such peptides are attracting attention as markers for disease in the field of clinical tests. For example, Simmonds' disease and Sheehan's syndrome caused by the reduced secretion of pituitary hormone can be clinically tested by detecting ACTH as a marker peptide. However, impurities contained in a biological sample to be used for the clinical test may have an influence on the detection of ACTH. Consequently, there is a demand for a method for detecting ACTH which can suppress the influence of impurities in the biological sample.
Document D1 (Livesey and Donald, 1982; Clinica Chimica Acta, 123 p. 193-198) addresses the problem of hormone adsorption to glass slides or plastic surfaces in radioimmunoassays for the detection of peptide hormones. In this document, it was reported that losses of hormone analyte in radioimmunoassays can most effectively be minimized by adding relatively large amounts of casein or derivatives thereof as opposed to the widely used albumin or Tween20, which do not always adequately prevent adsorption and hence must be tested for any particular application. - An object of the present invention is to provide uses of a material which can selectively adsorb ACTH as an adsorbent of ACTH. Another object of the present invention is to provide a method for detecting ACTH in a liquid sample using the novel material.
- The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
- Surprisingly, the present inventors have found out that a gel obtained from casein can selectively adsorb ACTH and completed the present invention.
According to a first aspect, the present invention provides: - (1) A method for detecting adrenocorticotropic hormone comprising:
- bringing a liquid sample containing adrenocorticotropic hormone into contact with gelled casein to adsorb adrenocorticotropic hormone to the gelled casein; and
- detecting the adrenocorticotropic hormone adsorbed to the gelled casein.
- (2) The method according to (1), wherein the adsorbing process of adrenocorticotropic hormone is performed by bringing the liquid sample into contact with the gelled casein in the range of pH 6.5 or more.
- (3) The method according to (1) or (2), further comprising:
- recovering the gelled casein brought into contact with the liquid sample; and
- allowing the adrenocorticotropic hormone adsorbed to the recovered gelled casein to be released.
- (4) The method according to (3), wherein the release process of adrenocorticotropic hormone is performed by bringing the recovered gelled casein into contact with an aqueous medium at a pH 5.5 or less and allowing the adrenocorticotropic hormone adsorbed to the gelled casein to be released into the aqueous medium.
- (5) The method according to any one of (1) to (4), wherein the gelled casein is casein gelled with a crosslinker.
- (6) The method according to (5), wherein the crosslinker is water-soluble carbodiimide.
- (7) The method according to any one of (1) to (6), wherein adrenocorticotropic hormone is a polypeptide having at least an amino acid sequence from the 1st to 24th position of adrenocorticotropic hormone.
- (8) The method according to any one of (1) to (7), wherein adrenocorticotropic hormone forms a complex with albumin.
- (9) The method according to any one of (1) to (8), wherein the liquid sample is blood, plasma, serum or urine.
- (10) According to a second aspect of the present invention, the use of gelled casein is provided as an adsorbent of adrenocorticotropic hormone.
- (11) The use according to (10), wherein the gelled casein is casein gelled with a crosslinker.
- (12) The use according to (11), wherein the crosslinker is water-soluble carbodiimide.
- (13) The use according to any one of (10) to (12), wherein adrenocorticotropic hormone is a polypeptide having at least an amino acid sequence from the 1st to 24th position of adrenocorticotropic hormone.
- (14) The use according to any one of (10) to (13), wherein adrenocorticotropic hormone forms a complex with albumin. In particular, the invention provides the use of gelled casein for the detection of adrenocorticotropic hormone by adsorbing adrenocorticotropic hormone to the gelled casein.
- The gelled casein contained in the adsorbent of ACTH of the present invention can selectively adsorb ACTH in the liquid sample. Therefore, the method for detecting ACTH of the present invention can detect ACTH in a state where influences of other impurities are limited by allowing ACTH in the liquid sample to be selectively adsorbed to the gelled casein.
-
-
Fig. 1 is a graph showing the adsorption capacity of gel carriers produced from various types of materials to ACTH; -
Fig. 2 is a graph showing the adsorption capacity of the gelled casein to various types of peptides; -
Fig. 3 is a graph showing that the gelled casein is adsorbed to the peptide portion in ACTH labeled with tetramethyl rhodamine (TMR); -
Fig. 4 shows a photograph when the ACTH adsorbed to the gelled casein is detected by SDS-PAGE and subsequent silver staining; -
Fig. 5 shows a photograph when the ACTH forming a complex with albumin, which is adsorbed to the gelled casein, is detected by SDS-PAGE and subsequent fluorescence imaging; -
Fig. 6 shows a photograph when the fact that the gelled casein is not adsorbed to albumin is confirmed by SDS-PAGE and subsequent silver staining; -
Fig. 7 is a graph showing that an affinity of the gelled casein for ACTH varies depending on the pH; -
Fig. 8 shows a photograph in which the fact that ACTH in the serum can be detected by the method for detecting ACTH of the present invention is shown by SDS-PAGE, subsequent silver staining, and fluorescence imaging; and -
Fig. 9 shows a photograph in which the fact that ACTH in the urine can be detected by the method for detecting ACTH of the present invention is shown by SDS-PAGE, subsequent silver staining, and fluorescence imaging. - In the method for detecting ACTH of the present invention (hereinafter also referred to "detection method"), a process of bringing a liquid sample containing ACTH into contact with gelled casein to allow ACTH to be adsorbed to the gelled casein is performed.
- ACTH (a hormone composed of 39 amino acids) is secreted from the pituitary gland in the brain and acts on the adrenal cortex to facilitate secretion of adrenocortical hormone.
- In the embodiment of the present invention, the ACTH being detected may be a natural ACTH produced in the living body, a peptide produced by cells such as mammalian cells into which the gene coding for ACTH is introduced, insect cells, Escherichia coli, or a peptide produced by chemical synthesis. Further, the ACTH has a high homology in amino acid sequence between biological species. Accordingly, the origin of ACTH is not particularly limited as long as it is a biological species that produces ACTH. Examples thereof include mammals (e.g., humans, mice, rats, dogs, and rabbits), birds (e.g., ostriches), and fish (e.g., smooth dogfish).
- In the embodiment of the present invention, the liquid sample is not particularly limited as long as it is a liquid sample which may contain ACTH. For example, it may be a biological sample. The biological sample includes body fluid. Preferable examples of the body fluid include blood, plasma, serum, and urine. When the liquid sample is any one of the biological samples, the sample may contain a substance capable of binding to ACTH, which is derived from the living body. In the embodiment of the present invention, ACTH in the liquid sample may form a complex with the substance capable of binding to ACTH. The substance which can be bound to ACTH includes albumin.
- The ACTH contained in the liquid sample may be the entire length of polypeptide or a fragment thereof. As a fragment of ACTH, a polypeptide having at least an amino acid sequence from the 1st to 24th position of ACTH is preferred.
- In the detection method of the present invention, the gelled casein which is used to adsorb ACTH can be produced by gelling casein. Here, the term "casein" means a phosphorus protein which is known as a main component of milk protein. It is known that the casein is composed of three major casein components: αs, β, and κ.
- In the embodiment of the present invention, the type of casein which is used as a raw material of the gelled casein is not particularly limited. Examples thereof include acid casein, casein sodium, αs-casein, β-casein, and κ-casein. The origin of casein is not particularly limited. Usable examples thereof include casein obtained from the milk of mammals such as cows and goats.
- The method for preparing gelled casein is not particularly limited as long as it can gelate casein. For example, it includes a method for preparing gelled casein by reacting a solution of casein with a crosslinker. As such a crosslinker, water-soluble carbodiimide (WSC, chemical name: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferable.
- When the WSC is used, the gelled casein can be obtained, for example, in the following manner. First, 0.1 to 20% (w/v) of a casein solution is mixed with 0.1 to 50% (w/v) of a WSC solution at a volume ratio of 1 : 0.2 to 50. The mixed solution thus obtained is heated at 65 to 200°C for 5 to 120 minutes to return to room temperature, resulting in formation of gelled casein in the mixed solution. Then, the gelled casein is recovered from the mixed solution by centrifugation, followed by washing with water or an appropriate buffer solution to obtain gelled casein.
- In the embodiment of the present invention, the form of the gelled casein is not particularly limited. Accordingly, the gelled casein obtained in the above manner may be suitably processed into a particle form, a membranous form or the like.
- In the embodiment of the present invention, the operation of bringing the gelled casein into contact with the liquid sample is not particularly limited. For example, the gelled casein may be added to the liquid sample to bring them into contact with each other, or a dispersion of the particulate gelled casein may be mixed with the liquid sample to bring them into contact with each other.
- As for conditions in the contact of the gelled casein with the liquid sample, from the viewpoint of improving the adsorbing efficiency of ACTH, the gelled casein is preferably brought into contact with the liquid sample under the conditions where the pH in the mixture of the gelled casein and the liquid sample is 6.5 or more, preferably from 6.5 to 10. This is based on the fact that the affinity of the gelled casein for ACTH varies depending on the pH, which has been found by the present inventors. A unit configured to adjust the pH is not particularly limited and it can be suitably selected from methods known in the art. For example, the pH can be adjusted by previously adding a buffer having a buffering action at a pH of 6.5 or more to the gelled casein and/or the liquid sample or adding the buffer to a mixture of the gelled casein and the liquid sample.
- In the contact of the gelled casein with the liquid sample, conditions other than the pH are not particularly limited. For example, the content ratio of the gelled casein to the liquid sample is from 1 : 2 to 1 : 1000 (at a volume ratio), preferably from 1 : 10 to 1 : 200. The ambient temperature is from 4 to 60°C, preferably from 4°C to room temperature, and the contact time is from 5 seconds to 60 minutes, preferably from 30 seconds to 1 minute.
- In the detection method of the present invention, the process of detecting the ACTH adsorbed to the gelled casein is performed after the contact of the gelled casein with the liquid sample.
- In the process, a unit configured to detect the ACTH adsorbed to the gelled casein is not particularly limited. For example, the gelled casein adsorbed to ACTH is recovered so that the ACTH adsorbed to the recovered gelled casein can be directly detected. Alternatively, the gelled casein adsorbed to ACTH is recovered and the ACTH is allowed to be released from the recovered gelled casein so that the released ACTH can be detected. That is, the detection method of the present invention may further include recovering the gelled casein brought into contact with the liquid sample and allowing the ACTH adsorbed to the recovered gelled casein to be released.
- A unit configured to recover the gelled casein may be suitably selected from units known in the art according to the form of the gelled casein used. For example, the gelled casein can be recovered by centrifuging the mixture of the gelled casein and the liquid sample and removing the supernatant.
- A unit configured to allow ACTH to be released from the gelled casein is not particularly limited. For example, ACTH can be eluted from the gelled casein by adding any protein denaturant known in the art to the gelled casein recovered. Examples of the protein denaturant include surfactants such as sodium dodecyl sulfate (SDS), urea, and guanidine.
- As described above, the present inventors have found the fact that the affinity of the gelled casein for ACTH varies depending on the pH. That is, in the embodiment of the present invention, the contact of the recovered gelled casein with an aqueous medium at a pH of 5.5 or less allows the ACTH adsorbed to the gelled casein to be released into the aqueous medium. A unit configured to adjust the pH is not particularly limited as long as it is a unit which does not prevent the subsequent detection of ACTH. It can be suitably selected from methods known in the art. The aqueous medium is not particularly limited. From the viewpoint of making the operation simpler, it is preferable to use an aqueous medium which can adjust the pH in the mixture of the gelled casein and the aqueous medium to 5.5 or less. Preferably usable examples of the aqueous medium include inorganic acids known in the art (e.g., dilute hydrochloric acid and dilute sulfuric acid), organic acids (e.g., acetic acid solutions), and buffer solutions having a buffering action at a pH of 5.5 or less (e.g., phosphate buffers and Good's buffers).
- A unit configured to detect the released ACTH is not particularly limited as long as it is a unit capable of detecting ACTH by distinguishing from other substances. It can be suitably selected from peptide detection methods known in the art. Examples of the detection unit include SDS-PAGE, subsequent gel staining or Western blotting, ELISA, chromatography, and mass spectrometry.
- As described above, in the embodiment of the present invention, it is possible that ACTH contained in the liquid sample is selectively adsorbed to the gelled casein, followed by release into an appropriate aqueous solvent. That is, the detection method of the present invention can be used when intending to selectively recover ACTH from a liquid sample and/or when intending to purify ACTH from a liquid sample. Therefore, the scope of the present invention includes a method for selectively recovering ACTH from a liquid sample and a method for purifying ACTH from a liquid sample.
- The adsorbent of ACTH for use according to the method of the present invention (hereinafter also referred to as "adsorbent") has a feature of containing the gelled casein and can be suitably used for the detection method of the present invention. That is, the ACTH in the liquid sample can be selectively adsorbed by using the adsorbent of the present invention, similarly to the gelled casein described in the detection method of the present invention. The ACTH to be adsorbed may be a polypeptide having at least an amino acid sequence from the 1st to 24th position of ACTH. Further, the ACTH may form a complex with a substance capable of binding to ACTH such as albumin.
- The form of the gelled casein contained in the adsorbent for use in the method of the present invention is not particularly limited. From the viewpoint of easy handling, the particle form is preferred. In this case, the adsorbent may be in the form of a dispersion prepared by dispersing in water or an appropriate buffer solution. Alternatively, the adsorbent may be in the form of a biuret reagent containing gelled casein particles dried by a known method such as freeze drying and water or an appropriate buffer solution for dispersing the particles at the time of usage. The concentration of the gelled casein particles in the dispersion can be suitably set by those skilled in the art. Usually, it is from 1 to 100 mg/ml, preferably from 5 to 50 mg/ml.
- The adsorbent for use in the method of the present invention may contain known additives in the art, in addition to the gelled casein. Examples of the additives include buffer solutions (e.g., phosphate buffers and Good's buffers), protein stabilizing agents (e.g., BSA), and antiseptics (e.g., sodium azide). In the present invention, the affinity of the gelled casein for ACTH changes according to the pH as described above. Thus, it is preferable to contain a buffer solution having a buffering action at a pH of 6.5 or more.
- The method for storing the gelled casein contained in the adsorbent for use in the method of the present invention is not particularly limited, and it may be stored at, for example, 4°C to room temperature.
- The adsorbent for use in the method of the present invention has a capacity of selectively adsorbing to ACTH as long as it contains the gelled casein produced in the same manner as described in the detection method of the present invention. The capacity can be confirmed by the detection method of the present invention. Alternatively, the adsorption capacity of ACTH may be confirmed as follows. First, a synthesized ACTH peptide solution with a known concentration is mixed with the produced adsorbent. The gelled casein contained in the adsorbent is recovered from the mixed solution thus obtained. The concentration of the synthesized ACTH peptide contained in the remaining solution is measured and the resulting concentration is compared with the concentration before mixing. As the result of comparison, if the concentration after mixing is sufficiently lower than that before mixing, it is determined that the produced adsorbent has a capacity as the adsorbent of the present invention.
- Hereinafter, the present invention will be described in detail with reference to Examples, however, the present invention is not limited thereto.
- Casein sodium (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in MilliQ (registered trademark) water (hereinafter simply referred to as "ultrapure water"; Millipore) to prepare a casein sodium solution (50 g/L (5%)). A 5% water-soluble carbodiimide (WSC, manufactured by DOJINDO LABORATORIES) solution (1 mL) and ultrapure water (3 mL) were added to the casein sodium solution (1 mL) to prepare a casein sodium-WSC mixed solution (5 mL) (final concentration of casein sodium: 1%, final concentration of WSC: 1%). The mixed solution was heated at 80°C for 10 minutes, followed by returning to room temperature to form a gel in the mixed solution. The supernatant was removed with a pipette from the solution containing the gel and the gelled casein was collected. Ultrapure water was added to the collected gelled casein. The mixture was centrifuged at 22200 G for 10 minutes using a centrifuge (CF15RXII, manufactured by Hitachi Koki Co., Ltd.) to allow it to be separated into a supernatant and gelled casein, followed by removal of the supernatant with pipette. The operation was repeated twice to wash the gelled casein. The gelled casein after washing was made into a fine gel carrier by vortexing and pipetting and the resulting carrier was dispersed in ultrapure water (5 mL). The obtained dispersion was a 1% gelled casein carrier dispersion.
- The same operation as the preparation of the gelled casein carrier dispersion was performed using bovine serum albumin (hereinafter referred to as "BSA": manufactured by Sigma Aldrich) in place of casein sodium to prepare a 1% albumin gel carrier dispersion.
- Gelatin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in ultrapure water and heated to 80°C to prepare a 10% gelatin solution (0.5 mL). The solution was cooled at 4°C for 2 hours to obtain a gelatin gel. Ultrapure water (4.5 mL) at 4°C was added to the obtained gelatin gel and the mixture was made into a fine gelatin gel carrier by pipetting. Then, a 1% gelatin gel carrier dispersion was prepared.
- The 1% gelled casein carrier dispersion (50 µL) prepared in (1) was mixed with a solution of a polypeptide having at least an amino acid sequence from the 1st to 24th position of ACTH (hereinafter referred to as "ACTH (1-24)" (300 µM, 5 µL, manufactured by Biologica Co.) which was labeled with tetramethyl rhodamine (TMR). A 1 M Bicine/NaOH buffer solution (pH 9) (10 µL) and ultrapure water (35 µL) were added to the mixture to prepare a mixed solution (100 µL). The final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of TMR-labeled ACTH (1-24) was adjusted to 15 µM. Similarly, the 1% albumin gel carrier dispersion in place of the 1% gelled casein carrier dispersion and the 1% gelatin gel carrier dispersion were used to prepare each mixed solution. The operation of the gelatin gel carrier was performed while cooling so that the gelatin gel was not dissolved in the solution.
- These mixtures were centrifuged at 22200 G for 3 minutes using the centrifuge to allow them to be separated into a supernatant and a precipitate. After the centrifugation, the fluorescence intensity of the supernatant at an excitation wavelength of 540 nm and a fluorescence wavelength of 580 nm was measured with a spectrophotofluorometer (F-7000, manufactured by Hitachi High-Technologies Corporation.). The amount of TMR remaining in the supernatant which was not adsorbed to each gel carrier, i.e., the amount of ACTH (1-24) was calculated. A 15 µM TMR-labeled ACTH (1-24) solution not containing the gel carrier was used as a reference. The results are shown in
Fig. 1 . - From
Fig. 1 , it was found that the gelled casein carrier had a high adsorption capacity to TMR-labeled ACTH (1-24) as compared with those of the albumin gel carrier and the gelatin gel carrier. - 10 µL of a 1000 µM ACTH (1-24) (manufactured by Biologica Co.) solution, 10 µL of a 1000 µM ITIH4 fragment (manufactured by Biologica Co.) solution, 10 µL of a 1000 µM Fibrinogen α fragment (manufactured by Biologica Co.) solution, and 10 µL of a 1000 µM FactorXIII fragment (manufactured by Biologica Co.) solution were mixed. The 1% gelled casein carrier dispersion (50 µL) prepared in Example 1 (1), a 1 M Bicine buffer solution (pH 9) (3 µL), and ultrapure water (7 µL) were added to the mixture to prepare a peptide mixed solution (100 µL). The final concentration of the gelled casein carrier in the peptide mixed solution was adjusted to 0.5% and the final concentration of each peptide was adjusted to 100 µM. The peptide mixed solution thus prepared was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate, and the supernatant was collected. Ultrapure water (99 µL) was added to the collected supernatant (1 µL) to prepare a 100-fold diluted supernatant. As an index sample for converting the MS concentration, a 1 µM TMR-labeled ACTH (1-24) solution (1 µL) was added to the supernatant to prepare a sample for measuring the peptide adsorption capacity. As a control for comparison with the sample for measuring the peptide adsorption capacity, a control sample was prepared by the same operation as described above using ultrapure water in place of the 1% gelled casein carrier dispersion.
- In advance, 10 mg/mL of αCHCA solution was prepared as a matrix solution. As a solvent, a solution of 0.1% trifluoroacetic acid/50% acetonitrile was used. Drops of the sample for measuring the peptide adsorption capacity (1 µL) prepared in (1) were put on an MS sample plate and dried under reduced pressure. Thereafter, drops of the matrix solution (1 µL) were put on the plate to be dried. It was confirmed that matrix crystals were formed. The MALDI-TOF/MS measurement (m/z of 800 to 5000) (Voyager-DE (trademark) PRO, manufactured by Applied Biosystems) was performed. The MALDI-TOF/MS measurement of the control sample was performed by the same operation as that of the MALDI-MS of the sample for measuring the peptide adsorption capacity using the control sample in place of the sample for measuring the peptide adsorption capacity. These measurement results were adjusted by the strength of the index sample to calculate the concentration of the peptides contained in the supernatants of each peptide. The results of the adsorption capacity of the gelled casein carrier to ACTH (1-24), ITIH4 fragment, Fibrinogen α fragment, and FactorXIII fragment are shown in
Fig. 2 . The properties of the peptides are shown in Table 1. InFig. 2 , the solid line of 100 µM shows the concentration of the peptides present in the peptide solution when the gelled casein carrier is not mixed.[Table 1] Peptide name The number of amino acid residues Isoelectric point Adsorption to gelled casein Amino acid sequence SEQ ID NO. ACTH(1-24) 24 10.6 + 1 ITIH4 fragment 21 12 - 2 Fibrinogen α fragment 25 6.3 - 3 Factor XIII fragment 25 3.9 - 4 - From
Fig. 2 , it was found that the adsorption capacity of the gelled casein carrier to ACTH (1-24) was significantly high as compared with those to ITIH4, Fibrinogen α, and Factor XIII. This showed that the gelled casein carrier did not adsorb ACTH (1-24) depending on isoelectric points such as acidic, neutral, and basic peptides, but selectively adsorbed ACTH (1-24). - A 1% gelled casein carrier dispersion was prepared in the same manner as described in (1) Preparation of gelled casein carrier dispersion of Example 1.
- The 1% gelled casein carrier dispersion (50 µL) prepared in (1) was mixed with a 300 µM TMR-labeled ACTH (1-24) solution (10 µL). Further, a 1 M Bicine buffer solution (pH 9) (10 µL) and ultrapure water (30 µL) were added thereto to prepare a mixed solution (100 µL). The final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of TMR-labeled ACTH (1-24) was adjusted to 30 µM. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate. As a control for comparison with a mixed solution of the gelled casein carrier and TMR-labeled ACTH (1-24), specifically a mixed solution not containing the gelled casein carrier, a mixed solution for comparison was also prepared by the same operation as described above using ultrapure water in place of the 1% gelled casein carrier dispersion.
- The same operation as the preparation of the gelled casein carrier and TMR-labeled ACTH (1-24) was performed using TMR in place of TMR-labeled ACTH (1-24) to prepare a mixed solution of the gelled casein carrier and TMR. As a control for comparison with a mixed solution of the gelled casein carrier and TMR, specifically a mixed solution not containing the gelled casein carrier, a mixed solution for comparison was also prepared by the same operation as described above using ultrapure water in place of the 1% gelled casein carrier dispersion.
- The fluorescence intensity of the supernatant at an excitation wavelength of 540 nm and a fluorescence wavelength of 580 nm in the mixed solution of the gelled casein carrier and TMR-labeled ACTH (1-24) prepared in (2) was measured with the spectrophotofluorometer (F-7000, manufactured by Hitachi High-Technologies Corporation.). The fluorescence intensity of the mixed solution not containing the gelled casein carrier prepared in (1) as a control was measured. The fluorescence intensity of the control was converted at 30 µM TMR-labeled ACTH (1-24) to calculate the amount of the TMR-labeled ACTH (1-24) which was not adsorbed to the gelled casein carrier and left in the supernatant.
- The fluorescence intensity of the supernatant at an excitation wavelength of 540 nm and a fluorescence wavelength of 580 nm in the mixed solution of the gelled casein carrier and TMR prepared in (2) was measured with the spectrophotofluorometer (F-7000, manufactured by Hitachi High-Technologies Corporation.). The fluorescence intensity of the mixed solution not containing the gelled casein carrier prepared in (1) as a control was measured. The fluorescence intensity of the control was converted at 30 µM TMR to calculate the amount of the TMR which was not adsorbed to the gelled casein carrier and left in the supernatant. Those results are shown in
Fig. 3 . - From
Fig. 3 , it is found that the gelled casein carrier hardly adsorbs TMR. This showed that the gelled casein carrier selectively adsorbed the portion of ACTH (1-24) in TMR-labeled ACTH (1-24). - A solution of 300 µM TMR-labeled ACTH (1-24) (10 µL), 1 M Bicine buffer solution (pH 9) (10 µL), and ultrapure water (80 µL) were mixed to prepare a mixed solution (100 µL). The mixed solution thus prepared was collected as a sample A.
- A 1 M Bicine buffer solution (pH 9) (10 µL) and ultrapure water (40 µL) were added to the 1% gelled casein carrier dispersion (50 µL) prepared in Example 1 to prepare a mixed solution (100 µL). The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate. The resulting precipitate was collected as a sample B.
- The 1% gelled casein carrier dispersion (50 µL) prepared in Example 1 was mixed with a solution (10 µL) of 300 µM TMR-labeled ACTH (1-24). A 1 M Bicine buffer solution (pH 9) (10 µL) and ultrapure water (30 µL) were added thereto to prepare a mixed solution (100 µL). The final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of ACTH (1-24) was adjusted to 30 µM. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate. The supernatant was collected as a sample C. Subsequently, ultrapure water (100 µL) was added to the precipitate, which was stirred with a vortex. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate. The precipitate was collected as a sample D in SDS-PAGE shown in
Fig. 4 . - In advance, NuPAGE LDS Sample Buffer (4x) (manufactured by Invitrogen) (50 µL), NuPAGE Sample Reduction Agent (10x) (manufactured by Invitrogen) (20 µL), and ultrapure water (30 µL) were mixed to prepare a 2-fold condensed sample buffer (100 µL). Ultrapure water (20 µL) and 2-fold condensed sample buffer (20 µL) were added to the sample D collected in (1), which was heated at 70°C for 10 minutes. After heating, the obtained sample was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a precipitate. The supernatant (10 µL) obtained by centrifugation was subjected to SDS-PAGE. The sample B collected in (1) was subjected to the same operation as that of the sample D and SDS-PAGE. Further, a 2-fold condensed sample buffer (5 µL) was added to 5 µL of the sample A collected in (1), which was heated at 70°C for 10 minutes. 10 µL of the resulting mixture was subjected to SDS-PAGE. The sample C collected in (1) was subjected to the same operation as that of the sample A and SDS-PAGE.
- As an electrophoresis apparatus for SDS-PAGE, a vertical mini electrophoresis system (manufactured by Invitrogen) was used. As electrophoresis gels, NuPAGE 4-12% Bis-Tris Gels (1.0 mm, 10 wells, manufactured by Invitrogen) were used. As a running buffer, a solution prepared by 20-fold dilution of NuPAGE MES SDS running buffer (20x) (manufactured by Invitrogen) was used. The voltage during electrophoresis was set to a constant value of 200 V. After SDS-PAGE, the gel was stained with silver using EzStain Silver (ATTO CORPORATION). The results are shown in
Fig. 4 . - From the results of
Fig. 4 , almost all the ACTH (1-24) is adsorbed to the gelled casein carrier and is not present in the supernatant. The ACTH (1-24) adsorbed to the gelled casein carrier could be detected by directly subjecting it to SDS-PAGE. Generally, it is known that an ACTH molecule becomes a polymer in the presence of base. Accordingly, the bands present on the TMR-ACTH are expected to be ACTH polymers. It was found that the gelled casein carrier adsorbed to the polymer of ACTH (1-24). - Casein sodium was dissolved in ultrapure water to prepare a 50 g/L (5%) casein sodium solution. A 5% water-soluble carbodiimide (WSC) solution (1 mL) and ultrapure water (3 mL) were added to the casein sodium solution (1 mL) to prepare a casein sodium-WSC mixed solution (5 mL) (final concentration of casein sodium: 1%, final concentration of WSC: 1%). The temperature of the mixed solution was returned to room temperature by heating at 80°C for 10 minutes and a gel was formed in the mixed solution. The liquid portion was removed from the mixed solution containing the gel with a pipette. A gel precipitate was collected as a gelled casein carrier. Ultrapure water was added to the collected gelled casein carrier. The resulting mixture was centrifuged at 22200 G for 10 minutes using the centrifuge to allow it to be separated into a supernatant and a gel carrier, followed by removal of the supernatant with a pipette. The operation was repeatedly performed twice to wash the gelled casein carrier. The gel carrier after washing was made into a fine gel carrier by vortexing and pipetting and the resulting carrier was dispersed in ultrapure water (3 mL). The dispersion was a 1.6% gelled casein carrier dispersion.
- A 2400 µM BSA solution (40 µL), 200 µM TMR-labeled ACTH (1-24) (8 µL), and ultrapure water (32 µL) were mixed to prepare a BSA-ACTH (1-24) complex solution (80 µL). The concentration of BSA in the solution is 1200 µM, and the concentration of TMR-labeled ACTH (1-24) was adjusted to 20 µM. The equilibrium dissociation constant (Kd) between BSA and TMR-labeled ACTH (1-24) was measured by the fluorescence titration method and the Kd value was 12 µM. That is, when the prepared complex solution is 2-fold diluted, the concentration of BSA is 600 µM. When the concentration of TMR-labeled ACTH (1-24) is 10 µM, 98.0% of TMR-labeled ACTH (1-24) forms a complex with BSA.
- The solution (80 µL) prepared in (2) was mixed with the 1.6% gelled casein carrier dispersion (50 µL) prepared in (1) . Thereafter, a 1 M Bicine/NaOH buffer solution (pH 9) (16 µL) and ultrapure water (14 µL) were added thereto to prepare a mixed solution (160 µL). The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The gelled casein carrier was subjected to the following operation (4). The supernatant was used as a "supernatant A" of a sample containing BSA. Further, a mixed solution not containing the gelled casein carrier, a BSA-ACTH (1-24) complex solution (80 µL), a 1 M Bicine/NaOH buffer solution (pH 9) (16 µL), and ultrapure water (64 µL) were added to prepare a mixed solution (160 µL) as a control. The solution was used as a reference (REF.) sample containing BSA.
- A mixed solution was prepared in the same manner as described in the preparation of mixed solution of gelled casein carrier and BSA-ACTH (1-24) complex using a 20 µM TMR-labeled ACTH (1-24) solution in place of the BSA-ACTH (1-24) complex solution. The gelled casein carrier was subjected to the following operation (4). The supernatant was used as a "supernatant A" of a sample not containing BSA. Further, a mixed solution not containing the gelled casein carrier, 20 mM TMR-labeled ACTH (1-24) (80 µL), a 1 M Bicine/NaOH buffer solution (pH 9, 16 µL), and ultrapure water (64 µL) were added to prepare a mixed solution (160 µL) as a control. The solution was used as a REF. sample not containing BSA.
- A 20 mM Bicine/NaOH buffer solution (160 µL) was added to the gelled casein carrier separated in (3) Preparation of mixed solution of gelled-casein carrier and BSA-ACTH (1-24) complex. The mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the resulting dispersion was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The gelled casein carrier was subjected to the following operation (5). The supernatant was used as a "supernatant B" of a sample containing BSA.
- A 20 mM Bicine/NaOH buffer solution (160 µL) was added to the gelled casein carrier separated in (3) Preparation of mixed solution of gelled-casein carrier and ACTH (1-24). The mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the resulting dispersion was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The gelled casein carrier was subjected to the following operation (5). The supernatant was used as a "supernatant B" of a sample not containing BSA.
- A 100 mM MES/NaOH buffer solution (pH 5) (160 µL) was added to the gelled casein carrier after the process in (4) Washing of gelled casein carrier mixed with BSA-ACTH (1-24) complex. The mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The supernatant was used as a "supernatant C" of a sample containing BSA.
- A 100 mMMES/NaOH buffer solution (pH 5) (160 µL) was added to the gelled casein carrier after the process in (4) Washing of gelled casein carrier mixed with ACTH (1-24). The mixture was pipetted so that the gelled casein carrier was uniformly dispersed in the solution. After pipetting, the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The supernatant was used as a "supernatant C" of a sample not containing BSA.
- In advance, a 10 x Loading Buffer (10 µL, manufactured by TAKARA), a 60% glycerol solution (5 µL), and ultrapure water (85 µL) were mixed to prepare a solution. The resulting solution was used as a sample buffer. Then, 20 µL of the REF. sample containing BSA, 20 µL of the sample supernatants A to C containing BSA, 20 µL of the REF. sample not containing BSA, and 20 µL of the sample supernatants A to C not containing BSA were collected. Each of the solutions was mixed with the sample buffer (2 µL), followed by SDS-PAGE. As an electrophoresis apparatus for SDS-PAGE, a vertical mini electrophoresis system (manufactured by Invitrogen) was used. As electrophoresis gels, NuPAGE 4-12% Bis-Tris Gels (1.0 mm, 10 wells, manufactured by Invitrogen) were used. As a running buffer, a solution prepared by 20-fold dilution of NuPAGE MES SDS running buffer (20x) (manufactured by Invitrogen) was used. The voltage during electrophoresis was set to a constant value of 200 V. Thus, after performing the SDS-PAGE, image data of electrophoresis gels (
Fig. 5 ) was obtained in the High Sample Intensity mode at the wavelength for TAMRA (excitation wavelength: 532 nm) of a fluorescence imager (Pharos FX (trademark) Molecular Imager: BIO-RAD LABORATORIES). Thereafter, the electrophoresis gels were stained with silver according to the procedure using EzStain Silver (ATTO CORPORATION) (Fig. 6 ). - The bands of TMR-labeled ACTH (1-24) can be confirmed from
Fig. 5 . The bands of BSA can be confirmed fromFig. 6 . In the mixed series (series containing BSA) prepared by mixing the gelled casein carrier and the BSA-ACTH (1-24) complex, the results of the REF. sample ofFig. 6 and the sample (the supernatant A) show that BSA is not adsorbed to the gelled casein carrier in the presence of the Bicine/NaOH buffer solution (pH 9). However, when smear bands of TMR-labeled ACTH (1-24) are confirmed inFig. 5 , the density of the bands is decreased. Thus, it is considered that a part of the TMR-ACTH (1-24) is detached from the BSA and adsorbed to the gelled casein carrier. It is surmised that the proportion in which the gelled casein carrier adsorbs TMR-ACTH (1-24) from BSA is determined by the balance between the equilibrium reaction of BSA and ACTH (1-24) and the equilibrium reaction of the gelled casein carrier and ACTH (1-24). In other words, in order to collect a larger amount of TMR-ACTH (1-24) from BSA, it is necessary to increase the additive amount of the gelled casein carrier. In the sample (the supernatant B), the band of TMR-labeled ACTH (1-24) can be hardly confirmed. Thus, it is expected that TMR-labeled ACTH (1-24) is still adsorbed to the gelled casein carrier in the washing process. In the sample (the supernatant C), the dark bands of TMR-labeled ACTH (1-24) can be confirmed. Accordingly, it was found that the TMR-labeled ACTH (1-24) was released from the gelled casein carrier in the presence of the MES/NaOH buffer solution (pH 5). - The above results suggested that even the ACTH (1-24) bound to BSA could be adsorbed to the gelled casein carrier. From the result, it was suggested that the ACTH (1-24) could be recovered by removing BSA from the BSA-ACTH (1-24) complex using the gelled casein.
- A 1% gelled casein carrier dispersion was prepared in the same manner as described in (1) Preparation of gelled casein carrier dispersion of Example 1.
- The 1% gelled casein carrier dispersion (50 µL) prepared in (1) was mixed with a 300 µM TMR-labeled ACTH (1-24) solution (10 µL), and a 1 M Bicine buffer solution (pH 9) (10 µL) and ultrapure water (30 µL) were added thereto to prepare a mixed solution (100 µL). The final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5% and the final concentration of TMR-labeled ACTH (1-24) was adjusted to 30 µM. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- A 20 mM Bicine/NaOH buffer solution (160 µL) was added to the gelled casein carrier separated in (2), followed by pipetting of the gelled casein carrier. After pipetting, the resulting dispersion was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier.
- Each solution with each pH shown in Table 2 was added to the gelled casein carrier washed in (3). The mixture was pipetted so that the gelled casein carrier was uniformly dispersed therein. After pipetting, the mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. In order to calculate the concentration of ACTH (1-24) remained in the separated supernatant, the fluorescence intensity of the supernatant was measured by the same operation as the spectrophotofluorometric measurement described in (3) of Example 3. As a control for comparison with the mixed solutions, a 30 µM TMR-labeled ACTH (1-24) solution containing each solution with each pH shown in Table 2 and not containing the gelled casein carrier was prepared. The fluorescence intensity of the solution was calculated in the same manner as described above. The fluorescence intensity of the control was converted at 30 µM TMR-labeled ACTH (1-24) to calculate the concentration of ACTH (1-24) remained in the supernatant from which the gelled casein carrier was removed. The measurement results are shown in Table 2 and
Fig. 7 .[Table 2] Solution pH Concentration of ACTH (1-24) in the supernatant [µM] (not containing the gelled casein carrier) Concentration of ACTH (1-24) in the supernatant [µM] (after dissociation from the gelled casein carrier) 100 mM Glycine/HCl buffer solution 2.5 30 27.8 Only HCl 2.5 30 31.1 Only HCl 3.9 30 25.2 10 mM acetic acid solution 4.1 30 30.0 30 mM MES solution 4.5 30 27.2 100 mM sodium acetate buffer solution 4.9 30 18.9 30 mM MES solution 4.9 30 25.7 100 mM MES/NaOH buffer solution 5.1 30 18.1 Only HCl 5.3 30 19.1 30 mM MES solution* 6 30 10.5 30 mM MES solution* 7.2 30 6.7 100 mM PBS buffer solution 7.3 30 1 Only HCl 7.7 30 0.8 30 mM ammonium bicarbonate solution 8.3 30 7.9 Only NaOH 8.4 30 1.1 100 mM Bicine/NaOH buffer solution 8.8 30 0.8 100 mM CAPS/NaOH buffer solution 10.3 30 3.2 * The pH of the 30 mM MES solution was adjusted with HCl and NaOH. - From Table 2 and
Fig. 7 , it was found that the ACTH (1-24) bound to the gelled casein carrier could be dissociated from the gelled casein carrier by using a solution having a pH of 5 or less regardless of the type of the solution. The 10 mM acetic acid solution and 30 mM ammonium bicarbonate solution used as pH adjustors are volatile. Thus, it is known that, in HPLC measurement or MS measurement, it is possible to perform a direct measurement without a process of demineralization. In other words, an appropriate solvent can be selected according to the measurement procedure of the solution after dissociation. For example, when ACTH (1-24) is detected by a biochemical procedure, a Glycine/NaOH buffer solution which is a Good's buffer or a MES/NaOH buffer solution may be used. In the case of a procedure which requires the process of demineralization before the treatment like the mass spectrometry, ACTH (1-24) can be dissociated using a volatile pH adjuster such as an acetic acid solution. - A 1% gelled casein carrier dispersion was prepared in the same manner as described in (1) Preparation of gelled casein carrier dispersion of Example 1.
- Serum previously collected from healthy woman (22 years old) (100 µL, PROMEDDX "Normal Serum Pool") was mixed with 300 µM TMR-labeled ACTH (1-24) (10 µL) to prepare serum containing TMR-labeled ACTH (1-24). The 1% gelled casein carrier dispersion (100 µL) prepared in (1) was placed in another container and centrifuged to allow the dispersion to be separated into a supernatant and a precipitate. The supernatant was removed with a pipette and only the precipitate was collected. Then, the serum containing TMR-labeled ACTH (1-24) prepared above (110 µL), a 1 M Bicine buffer solution (pH 9) (20 µL), and ultrapure water (20 µL) were added to the obtained precipitate to prepare a mixed solution (150 µL) of the gelled casein carrier and the sample. The final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.75%, the final concentration of TMR-labeled ACTH (1-24) was adjusted to 20 µM, and the dilution ratio of the serum was adjusted to 1.5-fold. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. As a reference sample before recovery not containing the gelled casein carrier, a mixed solution (150 µL) was prepared by adding serum containing the TMR-labeled ACTH (1-24) (110 µL), a 1 M Bicine buffer solution (pH 9) (20 µL), and ultrapure water (20 µL).
- A gelled casein carrier solution was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and serum containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the serum containing TMR-labeled ACTH (1-24). The solution was used as a control sample A. As a reference sample before recovery not containing the gelled casein carrier, a mixed solution (150 µL) was prepared by adding a 1 M Bicine buffer solution (pH 9) (20 µL) and ultrapure water (130 µL).
- A mixed solution of the gelled casein carrier and serum was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and serum containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the TMR-labeled ACTH (1-24). The solution was used as a control sample B. As a reference sample before recovery not containing the gelled casein carrier, a mixed solution (150 µL) was prepared by adding serum (100 µL), a 1 M Bicine buffer solution (pH 9) (20 µL), and ultrapure water (30 µL).
- The supernatant separated in (2) Preparation of mixed solution was removed with a pipette and the gelled casein carrier was collected. A 20 mMBicine/NaOH buffer solution (150 µL) was added to the gelled casein carrier, followed by pipetting of the gelled casein carrier. The resulting solution was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The operation was repeatedly performed twice. The same operation was performed on the controls A and B prepared in (2).
- A 100 mM MES/NaOH buffer solution (pH 5) (50 µL) was added to the gelled casein carrier after the process of (3) Washing of gelled casein carrier, followed by pipetting of the gelled casein carrier. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The supernatant was recovered with a pipette and it was used as a recovered sample. The same operation was performed on the samples of the controls A and B.
- In advance, NuPAGE LDS Sample Buffer (4x) (manufactured by Invitrogen) (50 µL), NuPAGE Sample Reduction Agent (10x) (manufactured by Invitrogen) (20 µL), and ultrapure water (30 µL) were mixed to prepare a 2-fold condensed sample buffer (100 µL). Ultrapure water (5 µL) and 2-fold condensed sample buffer (5 µL) were added to the reference sample before recovery which was collected in (2), and the resulting mixture was heated at 70°C for 10 minutes. Similarly, 2-fold condensed sample buffer (5 µL) was respectively added to the reference sample before recovery for the control samples A and B which were collected in (2), the sample after recovery which was collected in (4), and the sample after recovery for the control samples A and B, followed by heating at 70°C for 10 minutes to prepare samples. Each of the obtained samples (1 µL) was subjected to SDS-PAGE. The electrophoresis apparatus for SDS-PAGE, the electrophoresis gels, and the running buffer are the same as those of Example 4 (2). The voltage during electrophoresis was set to a constant value of 200 V. After SDS-PAGE, fluorescence images and silver-stained images were obtained by the same operation as that of Example 5 (6). The results are shown in
Fig. 8 . - From the results of
Fig. 8 , it was confirmed that the proteins in the serum were hardly adsorbed to the gelled casein carrier and ACTH (1-24) could be selectively recovered from the serum. - A 1% gelled casein carrier fluid concentrate was prepared in the same manner as described in (1) Preparation of gelled casein carrier fluid concentrate of Example 1 (1).
- Normal urine previously collected from a single donor (100 µL, ProMedDx) was mixed with 300 µM TMR-labeled ACTH (1-24) (10 µL) to prepare urine containing TMR-labeled ACTH (1-24). The 1% gelled casein carrier fluid concentrate (60 µL) prepared in (1) was placed in another container and centrifuged to allow the dispersion to be separated into a supernatant and a precipitate. The supernatant was removed with a pipette and only the precipitate was collected. Then, the urine (110 µL) containing TMR-labeled ACTH (1-24) prepared above and a 1 M Bicine buffer solution (pH 9) (10 µL) were added to the obtained precipitate to prepare a mixed solution (120 µL) of the gelled casein carrier and the sample. The final concentration of the gelled casein carrier in the mixed solution was adjusted to 0.5%, the final concentration of TMR-labeled ACTH (1-24) was adjusted to 25 µM, and the dilution rate of the urine was adjusted to 1.2-fold. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. As a reference sample before recovery not containing the gelled casein carrier, a mixed solution (120 µL) was prepared by adding the urine containing TMR-labeled ACTH (1-24) (110 µL) and a 1 M Bicine buffer solution (pH 9) (10 µL).
- A gelled casein carrier solution was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and urine containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the urine containing TMR-labeled ACTH (1-24). The solution was used as a control sample A. As a reference sample before recovery not containing the gelled casein carrier, a mixed solution (120 µL) was prepared by adding a 1 M Bicine buffer solution (pH 9) (10 µL) and ultrapure water (110 µL).
- A mixed solution of the gelled casein carrier and urine was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and urine containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of TMR-labeled ACTH (1-24). The solution was used as a control sample B. As a reference sample before recovery not containing the gelled casein carrier, a mixed solution (120 µL) was prepared by adding urine (100 µL), a 1 M Bicine buffer solution (pH 9) (10 µL), and ultrapure water (10 µL).
- A mixed solution of the gelled casein carrier and TMR-labeled ACTH (1-24) was prepared by the same operation as described in Preparation of mixed solution of gelled casein carrier and urine containing TMR-labeled ACTH (1-24) except that ultrapure water was used in place of the urine. The solution was used as a control sample C. As a reference sample before recovery not containing the gelled casein carrier, a mixed solution (120 µL) was prepared by adding 300 µM TMR-labeled ACTH (1-24) (10 µL), a 1 M Bicine buffer solution (pH 9) (10 µL), and ultrapure water (100 µL).
- The supernatant separated in (2) Preparation of mixed solution was removed with a pipette and the gelled casein carrier was collected. A 20 mM Bicine/NaOH buffer solution (120 µL) was added to the gelled casein carrier, followed by pipetting of the gelled casein carrier. The resulting solution was centrifuged at 22200 G for 5 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The operation was repeatedly performed twice. The same operation was performed on the control samples A to C prepared in (2).
- A 100 mM Glycine/NaOH buffer solution (pH 2.5) (60 µL) was added to the gelled casein carrier after the process of (3) Washing of gelled casein carrier, followed by pipetting of the gelled casein carrier. The mixed solution was centrifuged at 22200 G for 3 minutes using the centrifuge to allow it to be separated into a supernatant and a gelled casein carrier. The supernatant was recovered with a pipette and it was used as a recovered sample. The same operation was performed on the control samples A to C.
- In advance, NuPAGE LDS Sample Buffer (4x) (manufactured by Invitrogen) (50 µL), NuPAGE Sample Reduction Agent (10x) (manufactured by Invitrogen) (20 µL), and ultrapure water (30 µL) were mixed to prepare a 2-fold condensed sample buffer (100 µL). Ultrapure water (5 µL) and 2-fold condensed sample buffer (5 µL) were added to the reference sample before recovery which was collected in (2), and the resulting mixture was heated at 70°C for 10 minutes. Similarly, 2-fold condensed sample buffer (5 µL) was respectively added to the reference sample before recovery for the control samples A to C which were collected in (2), the sample after recovery which was collected in (4), and the sample after recovery for the control samples A to C, followed by heating at 70°C for 10 minutes to prepare samples. Each of the obtained samples (10 µL) was subjected to SDS-PAGE.
- The electrophoresis apparatus for SDS-PAGE, the electrophoresis gels, and the running buffer are the same as those of Example 4 (2). The voltage during electrophoresis was set to a constant value of 200 V. After SDS-PAGE, fluorescence images and silver-stained images were obtained by the same operation as that of Example 5 (6). The results are shown in
Fig. 9 . From the results ofFig. 9 , it was confirmed that the proteins in the urine were hardly adsorbed to the gelled casein carrier and ACTH (1-24) could be selectively recovered from the urine. -
- <110> SYSMEX CORPORATION
- <120> METHOD FOR DETECTING ADRENOCORTICOTROPIC HORMONE AND ADSORBENT
- <130> SYSM-069-EP
- <160> 4
- <170> PatentIn version 3.5
- <210> 1
<211> 24
<212> PRT
<213> Homo sapiens - <400> 1
- <210> 2
<211> 21
<212> PRT
<213> Homo sapiens - <400> 2
- <210> 3
<211> 25
<212> PRT
<213> Homo sapiens - <400> 3
- <210> 4
<211> 25
<212> PRT
<213> Homo sapiens - <400> 4
Claims (14)
- A method for detecting adrenocorticotropic hormone comprising:bringing a liquid sample containing adrenocorticotropic hormone into contact with gelled casein to adsorb adrenocorticotropic hormone to the gelled casein; anddetecting the adrenocorticotropic hormone adsorbed to the gelled casein.
- The method according to claim 1, wherein the adsorbing process of adrenocorticotropic hormone is performed by bringing the liquid sample into contact with the gelled casein in the range of pH 6.5 or more.
- The method according to claim 1 or 2, further comprising:recovering the gelled casein brought into contact with the liquid sample; andallowing the adrenocorticotropic hormone adsorbed to the recovered gelled casein to be released.
- The method according to claim 3, wherein the release process of adrenocorticotropic hormone is performed by bringing the recovered gelled casein into contact with an aqueous medium at a pH 5. 5 or less and allowing the adrenocorticotropic hormone adsorbed to the gelled casein to be released into the aqueous medium.
- The method according to any one of claims 1 to 4, wherein the gelled casein is casein gelled with a crosslinker.
- The method according to claim 5, wherein the crosslinker is water-soluble carbodiimide.
- The method according to any one of claims 1 to 6, wherein adrenocorticotropic hormone is a polypeptide having at least an amino acid sequence from the 1st to 24th position of adrenocorticotropic hormone.
- The method according to any one of claims 1 to 7, wherein adrenocorticotropic hormone forms a complex with albumin.
- The method according to any one of claims 1 to 8, wherein the liquid sample is blood, plasma, serum or urine.
- Use of gelled casein as an adsorbent of adrenocorticotropic hormone.
- Use according to claim 10, wherein the gelled casein is casein gelled with a crosslinker.
- Use according to claim 11, wherein the crosslinker is water-soluble carbodiimide.
- Use according to any one of claims 10 to 12, wherein adrenocorticotropic hormone is a polypeptide having at least an amino acid sequence from the 1st to 24th position of adrenocorticotropic hormone.
- Use according to any one of claims 10 to 13, wherein adrenocorticotropic hormone forms a complex with albumin.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2012005547A JP5717658B2 (en) | 2012-01-13 | 2012-01-13 | Method for detecting adrenocorticotropic hormone and adsorbent |
Publications (2)
Publication Number | Publication Date |
---|---|
EP2615103A1 EP2615103A1 (en) | 2013-07-17 |
EP2615103B1 true EP2615103B1 (en) | 2015-04-22 |
Family
ID=47471651
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP20130150698 Active EP2615103B1 (en) | 2012-01-13 | 2013-01-09 | Method for detecting adenocorticotropic hormone and absorbent |
Country Status (4)
Country | Link |
---|---|
US (1) | US9952228B2 (en) |
EP (1) | EP2615103B1 (en) |
JP (1) | JP5717658B2 (en) |
CN (1) | CN103207278B (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7242599B2 (en) * | 2020-03-17 | 2023-03-20 | 株式会社東芝 | MOLECULAR DETECTION DEVICE AND MOLECULAR DETECTION METHOD |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3758324A (en) * | 1971-12-23 | 1973-09-11 | Allied Chem | Cross linking agents for paper binders and paper coated therewith |
JPS60231167A (en) * | 1984-05-01 | 1985-11-16 | Nitsusui Seiyaku Kk | Diagnostic screening reagent and preparation thereof |
DE3640412A1 (en) * | 1986-11-26 | 1988-06-09 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING A SPECIFICALLY BINDABLE SUBSTANCE |
WO1997031626A1 (en) * | 1996-02-27 | 1997-09-04 | Teijin Limited | Powdery composition for nasal administration |
DE69817574T2 (en) * | 1997-07-02 | 2004-06-24 | Coloplast A/S | MANUFACTURING METHOD FOR A FIBER-FREE, POROUS MATERIAL |
SE0300791D0 (en) | 2003-03-20 | 2003-03-20 | Amersham Biosciences Ab | Use of ph-responsive polymers |
CN1776426A (en) * | 2005-11-29 | 2006-05-24 | 天津理工大学 | Semi-humid protein array and its preparing method |
US20070178308A1 (en) * | 2006-01-27 | 2007-08-02 | Konica Minolta Medical & Graphic, Inc. | Germanium nanoparticles and biosubstance labeling agent by use thereof |
EP2035830B1 (en) | 2006-06-14 | 2015-03-25 | Johns Hopkins University | Albumin-bound protein/peptide complex as a biomarker for disease |
US8268968B2 (en) * | 2006-12-13 | 2012-09-18 | Fujifilm Corporation | Method for producing modified biopolymer and method for crosslinking biopolymer |
EP2511707B1 (en) * | 2009-12-10 | 2017-02-01 | Sysmex Corporation | Method for detection of basic peptide and reagent for detection of basic peptide |
CN202033370U (en) * | 2011-04-06 | 2011-11-09 | 武汉优尔生科技股份有限公司 | Enzyme-linked immunosorbent assay kit of human adrenocorticotrophic hormone |
-
2012
- 2012-01-13 JP JP2012005547A patent/JP5717658B2/en active Active
-
2013
- 2013-01-09 EP EP20130150698 patent/EP2615103B1/en active Active
- 2013-01-11 US US13/739,723 patent/US9952228B2/en active Active
- 2013-01-11 CN CN201310010173.7A patent/CN103207278B/en active Active
Also Published As
Publication number | Publication date |
---|---|
US9952228B2 (en) | 2018-04-24 |
CN103207278B (en) | 2015-01-21 |
JP2013145172A (en) | 2013-07-25 |
US20130183762A1 (en) | 2013-07-18 |
EP2615103A1 (en) | 2013-07-17 |
CN103207278A (en) | 2013-07-17 |
JP5717658B2 (en) | 2015-05-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2023162207A (en) | Methods for rapid preparation of labeled glycosylamines and for analysis of glycosylated biomolecules producing the same | |
US20130236977A1 (en) | Compositions and methods for plasma peptide analysis | |
CN111801344A (en) | Nanopore protein conjugates for detection and analysis of analytes | |
JPH11503733A (en) | Method for purifying IGF-I | |
CN108948176B (en) | Osteopontin characteristic peptide and application thereof | |
CN111551730B (en) | Fluorescent microsphere sealing liquid and kit using same | |
EP4379377A1 (en) | Magnetic bead coating, preparation method therefor, and test kit | |
JP4106111B2 (en) | Calibration / control formulations for immunoassays | |
EP3564673A1 (en) | L-fabp immunoassay method and assay reagent used in said method | |
Cowan et al. | Procollagen type III amino‐terminal propeptide and insulin‐like growth factor I as biomarkers of growth hormone administration | |
EP2615103B1 (en) | Method for detecting adenocorticotropic hormone and absorbent | |
CN114705849A (en) | Mass spectrum method for absolute quantification of cystatin C in serum based on SILAC | |
KR20150125920A (en) | Method for preventing deterioration of unsensitized latex reagent | |
KR102386584B1 (en) | Methods and Systems for Determining ADAMTS13 Enzyme Activity | |
García-Murria et al. | Simple chemical tools to expand the range of proteomics applications | |
JP4039590B2 (en) | Stabilization of hemoglobin samples | |
US9448243B2 (en) | Weak affinity chromatography | |
JP6959883B2 (en) | Oxytocin quantification method | |
Giron et al. | Enrichment of N-terminal cysteinyl-peptides by covalent capture | |
EP3109630B1 (en) | Protein detection method using mass spectrometry | |
CN114705641B (en) | Determination reagent, kit and quantification method for chitinase 3-like protein 1 | |
US20070015911A1 (en) | Methods for biomarker discovery and diagnostic screening | |
CN111855861B (en) | Application of associated protein/peptide in improving proteome experiment efficiency | |
US20230204545A1 (en) | Kind of method for detecting sialoglycosyl casein glycomacropeptide | |
CN114705641A (en) | Chitinase3-like protein1 determination reagent, kit and quantitative method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
17P | Request for examination filed |
Effective date: 20131211 |
|
RBV | Designated contracting states (corrected) |
Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
17Q | First examination report despatched |
Effective date: 20140415 |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: TSURUOKA, RENA |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
INTG | Intention to grant announced |
Effective date: 20141211 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: REF Ref document number: 723171 Country of ref document: AT Kind code of ref document: T Effective date: 20150515 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602013001529 Country of ref document: DE Effective date: 20150603 |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: VDEP Effective date: 20150422 |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 723171 Country of ref document: AT Kind code of ref document: T Effective date: 20150422 |
|
REG | Reference to a national code |
Ref country code: LT Ref legal event code: MG4D |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150722 Ref country code: ES Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: PT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150824 Ref country code: HR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: LT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: RS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150723 Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: IS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150822 Ref country code: LV Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: PLFP Year of fee payment: 4 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 602013001529 Country of ref document: DE |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: EE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: DK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CZ Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: SK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: RO Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20150422 Ref country code: PL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
26N | No opposition filed |
Effective date: 20160125 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: BE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20160131 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20160109 Ref country code: BE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PL |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MC Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CH Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20160131 Ref country code: LI Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20160131 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: MM4A |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: PLFP Year of fee payment: 5 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20160109 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: PLFP Year of fee payment: 6 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CY Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: HU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO Effective date: 20130109 Ref country code: SM Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20160131 Ref country code: MK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 Ref country code: TR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: BG Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: AL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20150422 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20231130 Year of fee payment: 12 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20231212 Year of fee payment: 12 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: DE Payment date: 20231128 Year of fee payment: 12 |